Formation of biofilm in pathogenic bacteria defends them from antibiotics and the immune system of a host's life. Hence, investigation of the molecular mechanisms of biofilm formation and search for new substances counteracting this formation are becoming an attractive research area. In the course of our search for new inhibitors of biofilm formation in Mycobacterium species, we rediscovered a cyclic trihydroxamate siderophore, desferrioxamine E, from the culture of the marine-derived Actinomycete MS67. Desferrioxamine E inhibited biofilm formation of Mycobacterium smegmatis and M. bovis BACILLE de CALMETTE et GUÉRIN (BCG) with minimum inhibitory concentration (MIC) value of 10 µM, while no anti-microbial activity was observed up to 160 µM. Desferrioxamine E was also able to restore the anti-microbial activity of isoniazid against M. smegmatis by inhibiting biofilm formation. Mechanistic analysis of desferrioxamine E suggested that such inhibition might come from the depletion of iron in the medium, which is essential for biofilm formation in Mycobacterium species.